All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
Adenovirus derived recombinant vectors are attractive tools for gene transfer, including gene transfer into the central nervous system. First generation vectors (Ad) and high-capacity helper-dependent adenoviral vectors (HC-Ad) are the two main different types of vectors derived from adenovirus. Ad vectors are devoid of the essential E1a/1b genes, and are thus routinely grown in cell lines that express these genes in trans to allow adenoviral replication and packaging. HC-Ad genomes retain only cis-acting adenoviral sequences necessary to replicate the viral vector genomes (i.e., the inverted terminal repeats [ITRs] and the packaging signal sequence [ψ]). The absence of wild-type adenoviral sequences from HC-Ad genomes results in lower immunogenicity in vivo and promotes safer, efficient gene transfer with long lasting transgene expression. HC-Ads are grown with a helper virus that provides all essential adenoviral functions for replication in trans. The packaging sequence of the helper virus is flanked by either FRT (flippase [FLP] recombinase target sites) or loxP (Cre recombination targets sites), and thus HC-Ad are growing in either in 293-Flpe or 293-Cre cells. As the genomes replicate, the helper viral genome undergoes recombination that deletes the packaging site ψ; as a consequence, the helper virus genome is less efficiently packaged than the HC-Ad genome.
In spite of the early region gene deletions, and consequent lack of viral replication, first generation Ad vectors have residual expression of viral genes. This leads to a host adaptive immune response. Delivery of Ad vectors results in anti-capsid neutralizing antibodies that block re-infection with the same serotype of Ad vector. Also, injection with Ad vectors induces a CTL response directed against adenoviral proteins and the transgene.
Following systemic delivery of the vector, Ad capsid proteins activate chemokine expression from infected cells. The activation of innate responses by transcription-defective adenovirus particles has been demonstrated in mouse and nonhuman primate models. Serum IL-6, TNFalpha, IL-12 levels and liver toxicity occurred within hours in a dose dependent manner and were induced equally in animals receiving transcription competent or defective Ad vectors. Following intravenous administration, Ad vectors induce a biphasic course of cytokine and chemokine gene expression. The innate host defense system serves to rapidly eliminate Ad vectors, reducing transduction efficiency in vivo. Furthermore, at high titers, adenovirus vectors are associated with acute inflammation that may result in significant morbidity in transduced hosts. In the absence of viral transcription, it has been shown that the effects of the adenovirus particle do not extend beyond 24 hr.
After administration of Adenovirus-derived vectors in the Central Nervous System (CNS) transgene expression persists for long periods of time (i.e., 12 months). Injection of 1×106 to 1×107 iu of either first-generation or high capacity Ad into the brain cause a self-limiting and innate inflammatory reaction characterized by infiltration of macrophages and lymphocytes, increased expression of MHC class I, activation of local microglia and astrocytes localized to the injection site, and an increase in the expression of cytokine and chemokine genes (Byrnes et al. (1995). Adenovirus gene transfer causes inflammation in the brain. Neuroscience, 66(4):1015-24; Lowenstein (2000). Un pour tous, tous pour un. Trends Neurosci. 23(10):467-8; Zirger et al. (2006). Rapid upregulation of interferon-regulated and chemokine mRNAs upon injection of 108 international units, but not lower doses, of adenoviral vectors into the brain. J Virol. 80:5655-9.). Importantly, this initial innate inflammatory response is transient and does not reduce long term vector expression. However, acute adenovirus induced cytotoxicity is seen when vector doses of ≧108 iu are used to transduce the brain. These early innate inflammatory immune responses are caused by Ad vectors, but also by HC-Ad, or ultraviolet (UV)/psoralen-inactivated Ad; this confirms that viral genes are not necessary to stimulate innate immune responses; nevertheless, continued expression of viral antigens may be needed to stimulate an adaptive immune response against adenovirus, with an increase in neutralizing antibody titers and anti-Ad T cells. Furthermore, Barcia et al. showed novel helper-dependent high-capacity Ad sustain transgene expression for up to one year, even when injected into the brains of animals immunized against adenovirus preceding brain gene transfer. This strongly supports the use of HC-Ad for gene transfer into the brain, not only for short term gene expression, but also for long term gene expression, and potentially for gene therapy for human neurological diseases. Further, the incapacity of HC-Ad to induce systemic antiadenoviral immune responses further supports the safety and potential efficacy of these vectors (Barcia et al. (2007). Sustained, one year expression from high-capacity helper-dependent adenoviral vectors delivered to the brain of animals with a pre-existing systemic anti-adenoviral immune response: implications for clinical trails. In press).
While injection of first generation Ad vectors into the brain parenchyma causes acute cellular- and cytokine-mediated inflammatory responses, this does not affect transgene expression and it is dose dependent. In the presence of adenoviral immune responses, transgene expression from first generation adenovirus is rapidly ablated. Adenovirus induced cytotoxicity is only seen when high vector doses of greater than 108 i.u. are used to transduce the target tissue.
An important issue in gene therapy is how to improve the overall efficiency of gene delivery. Increasing transgene expression per vector genome delivered is one method for achieving this aim. An approach to do so is through the use of sequences that either increase the number of transcript copies, or reduce the turnover of the mRNA; both approaches would achieve higher level of protein being expressed per vector particle. Recently, the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) has been utilized in gene transfer vectors to enhance transgene expression. Enhanced transgene expression in adenovirus vectors, adeno-associated virus vectors, lentivirus vectors and MLV-derived vectors harboring WPRE has been reported (Loeb et al. (1999). Enhanced expression of transgenes from adeno-associated virus vectors with the woodchuck hepatitis virus posttranscriptional regulatory element: implications for gene therapy. Hum Gene Ther. 10:2295-305; Zufferey et al. (1999). Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors. J Virol. 73:2886-92; Glover et al. (2002). Adenoviral-mediated, high-level, cell-specific transgene expression: a SYN1-WPRE cassette mediates increased transgene expression with no loss of neuron specificity. Mol Ther 5:509-16; Ketteler et al. (2002). Enhanced transgene expression in primitive hematopoietic progenitor cells and embryonic stem cells efficiently transduced by optimized retroviral hybrid vectors. Gene Ther. 9:477-87; Mautino et al. (2002). Enhanced inhibition of human immunodeficiency virus type 1 replication by novel lentiviral vectors expressing human immunodeficiency virus type 1 envelope antisense RNA. Hum Gene Ther. 13:1027-37; Xu et al. (2003). Woodchuck hepatitis virus post-transcriptional regulation element enhances transgene expression from adenovirus vectors. Biochim Biophys Acta. 1621:266-71).
The inventors have previously demonstrated long term expression of Herpes virus type 1 Thymidine kinase (HSV1-TK) in experiments in which Ad-expressing HSV1-TK had been used in a paradigm of gene therapy for the treatment of rat glioblastoma. The inventors found high level, anatomically widespread and long term expression of HSV-1-TK (Dewey et al. (1999). Chronic brain inflammation and persistent herpes simplex virus 1 thymidine kinase expression in survivors of syngeneic glioma treated by adenovirus-mediated gene therapy: implications for clinical trials. Nat Med. 5:1256-63). If a second transgene was encoded by a second vector, co-injected with Ad-TK, no changes were seen in the expression of β-galactosidase, suggesting the hypothesis of that the observed effects were to HSV1-TK (Zermansky et al. (2001). Towards global and long-term neurological gene therapy: unexpected transgene dependent, high-level, and widespread distribution of HSV-1 thymidine kinase throughout the CNS, Mol Ther. 4(5): 490-8).
However, it was recently shown that HSV1-TK sequences could restitute expression to genes that had been made intronless, where the expression is strictly intron-dependent. HSV1-TK sequences fused to the 5′ end of a highly intron-dependent β-globin (Liu et al. (1995). HnRNP L binds a cis-acting RNA sequence element that enables intron-dependent gene expression. Genes Dev. 9:1766-80; Otero et al. (1998). Splicing-independent expression of the herpes simplex virus type 1 thymidine kinase gene is mediated by three cis-acting RNA subelements. J Virol. 72:9889-96), or fused to the 3′ end of the Hepatitis virus B surface antigen gene has been shown to provide expression in a intron-independent manner.
High doses of adenoviral vectors delivered to the brain have been demonstrated to induce adaptive immune responses that mediate detrimental side effects in clinical models. Enhancement of transgene expression constitutes an important shortcoming in an Ad-based therapy.
In light of the value of recombinant vectors for gene transfer and delivery and the deficiency of the overall efficiency of gene delivery, there exists a need in the art for improvements in the overall efficiency of gene delivery.